In order to develop a protective carrier scaffolder for the external usage of medical and hygienic materials, three essential protective elements existing in nature, i.e., algin, cellulose, and calcium phosphate apatite, were investigated. The algin is a main skeletal component of sea weeds, the cellulose is of vegetables, and the calcium phosphate apatite is of vertebral animals. In the present study we select the agarose which is a derivative from algin, the cellulose fiber obtained from skin of sea squirt, calcium oxide purified from shell powder, and tricalcium phosphate apatite purchased commercially. Consequently, the agarose-cellulose hybrid was made by the hydrogen bonds intermediating the calcium phosphate apatite between agarose and cellulose molecules. As the calcium phosphate apatite is formed by the addition of calcium hydroxide into tricalcium phosphate solution, we used calcium oxide to accelerate the hybridization between the agarose and calcium phosphate apatite and also between the cellulose and calcium phosphate apatite. In the phase contrast microscopic observation the agarose-cellulose hybrid showed more compact matrix structure than the mixture of agarose and cellulose. The agarose-cellulose hybrid showed increased storage modulus but decreased loss modulus in Rheometer test compared to those of the other materials tested in this study, representing that the agarose-cellulose hybrid has the highest elasticity among them and similar water capacity to agarose. The agarose-cellulose hybrid showed the strongest antimicrobial effect in bacteria killing assay than the other materials, and also it showed a potent blood clotting effect but no immunological hypersensitivity on the human skin. From the above results we presumed that the nobel material, agarose-cellulose hybrid, is a compact scaffolding matrix which has proper elasticity, high capacity to hold substrates, and antimicrobial and blood clotting property potent enough to carry the bio-medical and hygienic materials for external treatment safely.

In order to perform the protein analysis using the paraffin sections previous fixed with formalin, we applied the ImmunoMemBlot (IMB) method1) to detect the epitopes of target proteins with specific antibodies. In this study the protein extracts were obtained from the paraffin sections of each representative case of ameloblastoma, adenomatoid odontogenic tumor (AOT), and normal gingiva, and more a protein extract from fresh tissue of ameloblastoma was also compared to evaluate the IMB results used with 24 different antibodies. First of all, in the comparison between the paraffin section extract and fresh tissue extract of ameloblastoma, the latter consistently showed more positive IMB reaction than the former. Meanwhile, the paraffin section extract of ameloblastoma was more comparable with that of normal gingival, disclosing that most of proliferating genes, oncogenes, and apoptosis related genes, i.e., PCNA, CDK4, c-erbB2, CEA, p53, Bax, Bad, FLIP, FAS, Bcl-2, p21, N-ras, MMP-2, MMP-9, caspase-3, -8, -9, were highly expressed in ameloblastoma, but EGFR, HGF, and VEGF were similarly expressed both in the ameloblastoma and in normal human gingiva. On the other hand, the comparison between ameloblastoma and AOT both in the immunohistochemistry and IMB using their paraffin section extracts clearly demonstrated that the ameloblastoma showed more expression of proliferating genes and oncogenes while the AOT showed more expression of apoptosis related genes, i.e., Bax, Bad, FLIP, and caspase-9. Taken together, these data suggest that the IMB can be used for the primary screening of quantitative protein analysis using the paraffin section extract, and that the IMB results could be evaluated in conjunction with the immunohistochemical observation.

Immuno-MemBlot is a technique for detecting, analyzing, and identifying proteins, similar to the Western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular or slot templates directly onto the membrane. Recently we developed a new Immuno-MemBlot (IMB) method applying immunoreactions and coloring procedures directly in the wells of MemBlot apparatus, which were connected by canals to perform drainage for reagent application and buffer irrigation. This IMB method was designed to get theimmunoblot results more rapidly and clearly than the previous immunoblot ones. This study is aimed to evaluate the analytical accuracy of IMB using different biological assay. In the sensitivity test of IMB the monoclonal antibody can clearly detect the 30 ng (about 12 pM) of Mucocidin peptide (35 mer), and is also available to detect at least 10 ng (about 4 pM) of Mucocidin peptide (35 mer). The IMB was effective in the quantitative analysis of methothrexate (MTX) assay for cellular apoptosis. And more, this IMB is useful to screen large number of specific samples with ease and accuracy in a short time. In the screenings for the presence of Mucocidin in saliva the quantitative comparison is conspicuous among 48 persons depend on the different conditions ofgender, drinking and smoking habits, and oral diseases. Therefore, it is presumed that, even though the target proteins were partly degraded, a specific epitope can be detected if a monoclonal antibody was still reactive. Conclusively, these data suggest that the IMB can be useful in the primary qualitativeand quantitative analysis of proteins in various fluids, i.e., blood, saliva, tear, urine, etc.